As used herein, the term "blood products" includes whole blood and cellular components derived from blood, including erythrocytes (red blood cells) and platelets.
There are more than thirty blood group systems, one of the most important of which is the ABO system. This system is based on the presence or absence of antigens A and/or B. Blood of group A contains antigen A on its erythrocytes. Similarly, blood of group B contains antigen B on its erythrocytes. Blood of group AB contains both antigens, and blood of group O contains neither antigen. These antigens are found on the surface of erythrocytes, which are red blood cells containing hemoglobin, the principal function of which is the transport of oxygen.
This invention is directed to the purification of an enzyme capable of removing the A antigens from the surface of cells in the blood products. There are three recognized major sub-types of blood type A. These sub-types are known as A.sub.1, A intermediate (A.sub.int) and A.sub.2. There are both quantitative and qualitative differences which distinguish these three sub-types. Quantitatively, A.sub.1 erythrocytes have more antigenic A sites, i.e., terminal N-acetyl-galactosamine residues, than A.sub.int erythrocytes which in turn have more antigenic A sites than A.sub.2 erythrocytes. Qualitatively, the transferase enzymes responsible for the formation of A antigens differ biochemically from each other in A.sub.1, A.sub.int and A.sub.2 individuals. Some A antigens found in A.sub.1 cells contain dual A antigenic sites. All three sub-type antigens must be removed in order to convert blood products from type A to type O thereby rendering the blood products universal for transfusion therapy.
Blood of group A contains antibodies to antigen B. Conversely, blood of group B contains antibodies to antigen A. Blood of group AB has neither antibody, and blood group O has both. A person whose blood contains either (or both) of the anti-A or anti-B antibodies cannot receive a transfusion of blood containing the corresponding incompatible antigen(s). If a person receives a transfusion of blood of an incompatible group, the blood transfusion recipient's antibodies coat the red blood cells of the transfused incompatible group and cause the transfused red blood cells to agglutinate, or stick together. Transfusion reactions and/or hemolysis (the destruction of red blood cells) may result therefrom.
In order to avoid red blood cell agglutination, transfusion reactions and hemolysis, transfusion blood type is cross-matched against the blood type of the transfusion recipient. For example, a blood type A recipient can be safely transfused with type A blood which contains compatible antigens. Because type O blood contains no A or B antigens, it can be transfused into any recipient with any blood type, i.e., recipients with blood types A, B, AB or O. Thus, type O blood is considered "universal", and may be used for all transfusions. Hence, it is desirable for blood banks to maintain large quantities of type O blood. However, there is a paucity of blood type O donors. Therefore, it is useful to convert types A, B and AB blood to type O blood in order to maintain large quantities of universal blood products.
In an attempt to increase the supply of type O blood, methods have been developed for converting types A, B and AB blood to type O blood. For example, U.S. Pat. No. 4,609,627 entitled "Enzymatic Conversion of Certain Sub-Type A and AB Erythrocytes" ("the '627 Patent"), is directed to a process for converting A.sub.int and A.sub.2 (including A.sub.2 B erythrocytes) to erythrocytes of the H antigen type, as well as to compositions of type B erythrocytes which lack A antigens, which compositions, prior to treatment, contained both A and B antigens on the surface of said erythrocytes. The process for converting A.sub.int and A.sub.2 erythrocytes to erythrocytes of the H antigen type, which is described in the '627 Patent, includes the steps of equilibrating AB erythrocytes, contacting the equilibrated erythrocytes with .alpha.-N-acetylgalactosaminidase enzyme for a period sufficient to convert the A antigen to the H antigen, removing the enzyme from the erythrocytes and reequilibrating the erythrocytes.
The '627 Patent also discloses a method for purifying the .alpha.-N-acetylgalactosaminidase enzyme utilized in the conversion process. The disclosed purification procedure is extremely complicated and time-consuming and requires, in addition to several other steps, the extraction with acetone or the freeze drying of trimmed chicken livers in order to dehydrate the chicken livers. The procedure also requires the use of (1) a linear gradient to elute a protein fraction containing .alpha.-N-acetylgalactosaminidase, (2) two anion exchange columns, and (3) a BioGel P-150 column.
Further problems with the enzyme purification procedure described in the '627 Patent are that the procedure does not lend itself to large scale production. Hence, a need has arisen to develop a procedure for purifying enzymes which may then be capable of removing A antigens from the surface of cells in blood products which procedure is simple, efficient, lends itself to large scale use and produces an enzyme which is purer and has a higher specific activity.
In Levy et al., Journal of Biological Chemistry,Vol. 255, No. 24, dated Dec. 25, 1980, pages 11737-11742, isolation of .alpha.-N-acetylgalactosaminidase from Clostridium perfringens bacteria is described. However, the isolated bacterial .alpha.-N-acetylgalactosaminidase enzyme is not fully purified from contaminating sialidase, .beta.-galactosidase, and .beta.-N-acetylgalactosaminidase enzymes, making this preparation unsuitable for use in treating blood products for transfusion. Further, the bacterial enzyme described by Levy et al. achieved only a partial removal of A antigenicity, as evidenced by the fact that the cells treated with the bacterial enzyme agglutinated with human anti-A antiserum.
In "Glycosidases of Ehrlich Ascites Tumor Cells and Ascitic Fluid-Purification and Substrate Specificity of .alpha.-N-Acetylgalactosaminidase and .alpha.-Galactosidase: Comparison with Coffee Bean .alpha.-Galactosidase", by Yagi et al. in Archives of Biochemistry and Biophysics, Vol. 280, No. 1, dated July 1990, pages 61-67, the enzymes .alpha.-N-acetylgalactosaminidase and .alpha.-galactosidase were isolated from Ehrlich ascites tumor cells on .epsilon.-aminocaproylgalactosylamine-Sepharose. Again, the ascites .alpha.-N-acetylgalactosaminidase enzyme purified by Yagi et al. is not pure, as the enzyme shows more than one band on PAGE analysis. As stated on page 63 of the article, "neither the .alpha.-galactosidase nor the .alpha.-N-acetylgalactosaminidase [purified from Ehrlich ascite tumor cells] were homogeneous on polyacrylamide gel electrophoresis."
In addition, U.S. Pat. Nos. 4,427,777 and 4,330,619, as well as the article "Single-Unit Transfusions of RBC Enzymatically Converted from Group B to Group O to A and O Normal Volunteers", by Lenny et al. in Blood, Vol. 77, No. 6, Mar. 15, 1991, pages 1383-1388 are all directed to the purification and use of .alpha.-galactosidase enzyme for the removal of B antigens from erythrocytes. The paper entitled "Affinity Purification of .alpha.-galactosidase A from Human Spleen, Placenta and Plasma with Elimination of Pyrogen Contamination", by Bishop et al., in the Journal of Biological Chemistry, Vol. 286, No. 3, dated Feb. 10, 1981, pages 1307-1316 is directed to purification of .alpha.-galactosidase enzyme. However, to date, no method for purifying the enzyme .alpha.-N-acetylgalactosaminidase to homogeneity has been developed.
It is therefore an object of this invention to provide an improved method for purifying the enzyme .alpha.-N-acetylgalactosaminidase, which enzyme is capable of removing A antigens from the surface of cells in blood products.
It is another object of this invention to provide an improved method for purifying an enzyme capable of removing A antigens from the surface of cells in blood products, which method is simple and efficient, and can be used for large scale purification of said enzyme.
It is still another object of this invention to provide an enzyme capable of removing A antigens from the surface of cells in blood products wherein said enzyme is pure and homogeneous on polyacrylamide gel electrophoresis.
It is a further object of this invention to provide an enzyme capable of removing A antigens from the surface of cells in blood products, wherein said enzyme has a higher specific activity than enzymes previously purified.